Clinical Validation and Implementation of a Targeted Next-Generation Sequencing Assay to Detect Somatic Variants in Non-Small Cell Lung, Melanoma, and Gastrointestinal Malignancies

Abstract

We tested and clinically validated a targeted next-generation sequencing (NGS) mutation panel using 80 formalin-fixed, paraffin-embedded (FFPE) tumor samples. Forty non-small cell lung carcinoma (NSCLC), 30 melanoma, and 30 gastrointestinal (12 colonic, 10 gastric, and 8 pancreatic adenocarcinoma) FFPE samples were selected from laboratory archives. After appropriate specimen and nucleic acid quality control, 80 NGS libraries were prepared using the Illumina TruSight tumor (TST) kit and sequenced on the Illumina MiSeq. Sequence alignment, variant calling, and sequencing quality control were performed using vendor software and laboratory-developed analysis workflows. TST generated !500Â coverage for 98.4% of the 13,952 targeted bases. Reproducible and accurate variant calling was achieved at !5% variant allele frequency with 8 to 12 multiplexed samples per MiSeq flow cell. TST detected 112 variants overall, and confirmed all known single-nucleotide variants (n Z 27), deletions (n Z 5), insertions (n Z 3), and multinucleotide variants (n Z 3). TST detected at least one variant in 85.0% (68/80), and two or more variants in 36.2% (29/80), of samples. TP53 was the most frequently mutated gene in NSCLC (13 variants; 13/32 samples), gastrointestinal malignancies (15 variants; 13/25 samples), and overall (30 variants; 28/ 80 samples). BRAF mutations were most common in melanoma (nine variants; 9/23 samples). Clinically relevant NGS data can be obtained from routine clinical FFPE solid tumor specimens using TST, benchtop instruments, and vendor-supplied bioinformatics pipelines. (J Mol Diagn 2016, 18: 299e315; http:// dx

Clinical framework for next generation sequencing based analysis of treatment predictive mutations and multiplexed gene fusion detection in non-small cell lung cancer

“…In agreement with both Fisher et al [5] and Hagemann et al [2], the success rate and clinical feasibility of our NGS framework is highly dependent on central pathological review by experienced diagnostic pathologists together with standardized and quality controlled tissue handling, to ensure sufficiently high proportions of malignant cells in specimens with adequate nucleic acid quality. A challenge for regional/county hospitals may be the bioinformatics aspect of NGS.…”

Targeting adhesion signaling in KRAS, LKB1 mutant lung adenocarcinoma

“…We investigated whether our results could be applied to identify KRAS and LKB1 mutant lung adenocarcinoma patients who may benefit from FAK pathway inhibition. We used a targeted and clinically validated next-generation sequencing (NGS) mutation panel (22) to assay formalin-fixed, paraffin-embedded (FFPE) patient tissue to identify KRAS and KRAS and LKB1 mutant lung adenocarcinoma. Tissue sections from 12 total patients (n = 6 KRAS and n = 6 KRAS and LKB1) were assayed by confocal microscopy for E-Cad expression, and identical fields were then imaged via SHG to visualize collagen.…”

Pilot study of clinical targeted next generation sequencing for prostate cancer: Consequences for treatment and genetic counseling.

“…In prostate cancer there is not yet routine use of tNGS testing, in part because targets linked to treatment options have not yet been as well-defined and validated as in colorectal cancer, lung cancer, and melanoma [2,3]. We conducted this pilot study to assess the feasibility and utility of integrating a tNGS in the setting of a real-world prostate cancer clinical practice using the OncoPlex platform.…”

“…Targeted next generation sequencing DNA assays (tNGS) can identify actionable mutations in DNA from tumors that associate with responses to targeted therapeutics and are used in routine clinical practice in the management of lung cancer, colorectal cancer and melanoma [2,3]. Although tNGS is not yet in widespread clinical use for prostate cancer, largely due to the paucity of well-defined targets predictive of drug sensitivity, a number of recent studies have indicated potential actionable targets, most notably platinum chemotherapy and poly-ADP ribose polymerase inhibitors (PARPi) for tumors with DNA repair defects [4][5][6][7][8].…”